The Potential of Nitrogen Rich Surfaces to Distinguish between Cells of the Nucleus Pulposus and Annulus Fibrosus

Abstract

Introduction Mesenchymal stem cells (MSCs) have been proposed for the purposes of tissue engineering and repair of intervertebral disk (IVD) tissue. These cells would be induced to differentiate into cells with a disc-cell like phenotype, as an alternative treatment for against disk degeneration. One major drawback is that there currently exists no simple way to distinguish a nucleus pulposus (NP) cell from its annulus fibrosus (AF) or hyaline cartilage counterparts. In addition, immature NP cells produce similar macromolecules to those of hyaline cartilage1; therefore, it is difficult to confirm MSCs differentiation into a disk cell phenotype. Our previous studies have shown that surfaces rich in primary amines, -NH2, can influence the adhesion properties of cells.2 In this study, we created reproducible NH2-rich surfaces by low-pressure plasma polymerization of ethylene-ammonia mixture (so-called “L-PPE: N”) to test the hypothesis that selective adhesion of disk cells can be used to distinguish between an NP and an AF phenotype. This would serve as a marker to distinguish an appropriate NP, generated by tissue engineering techniques, from other cartilaginous phenotypes that may be inappropriate for function in the disc. Materials and Methods Surface preparation L-PPE: N coatings approximately 100 nm thick were prepared as previously described.3 These deposits, which contain about 7.5% NH2, were deposited on poly(ethylene terephthalate) (PET) film in a low-pressure (L) capacitively coupled radio-frequency glow-discharge plasma reactor. However, half of the films' surfaces were masked, so as to remain uncoated, bare PET. Cell isolation and culture Adult bovine tails (2 to 3 years old) were obtained 2 hours after slaughter at a local abattoir. The IVDs were dissected from their adjacent vertebral bodies and separated into nucleus pulposus (NP) and AF. Both tissues of the AF and the NP were first weighed (wet weight) and then subjected to a pronase digestion (0.2% W/V), followed by a digestion of collagenase 1A (0.04% W/V) for NP tissue and collagenase II (0.04% W/V) for AF tissue. After isolation, the AF cells and the NP cells were separately cultured on the half-coated surfaces in DMEM high glucose, supplemented with 10% FBS and 1% penicillin/streptomycin in a concentration of 1 × 106 cells/mL. Regular polystyrene culture dishes (TCPS) were used as a control. After 4 days of culture, cells were washed with ddH20, fixed and stained with Safranin-O. Images were captured of both cell types before and after wash, using an optical microscope. Results AF cells adhered preferentially to the L-PPE: N-coated surface, but did not adhere to the bare part of the PET surface (Fig. 1). NP cells, however, indiscriminately adhered to both PET and L-PPE: N surfaces. Both NP and AF cells adhered to regular TCPS dishes after 4 days. After washing with ddH20, which was meant to remove any nonadherent cells, and to provide a clearer image to be captured, AF cells remained bound to L-PPE:N surfaces; however, NP cells were completely removed from both surfaces. Conclusion In this study, we show that AF cells adhered preferentially to the substrate's portion that was L-PPE: N-coated, whereas NP cells did not manifest any preference to coated or uncoated portions. This preferential attachment to an amine-containing surface can be used to distinguish NP from AF cells; together with the production of a high GAG-to-hydroxyproline (proteoglycan-to-collagen) ratio by NP cells, it could help in identifying an NP-like phenotype. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Mwale F, et al. European Cells and Materials 2004;8:58–64 Girard-Lauriault PL, et al. Macromolecular Bioscience 2009;9:911–921 Girard-Lauriault PL, et al. Plasma Processes and Polymers 2005;2:263–270